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Eur J Appl Physiol
DOI 10.1007/s00421-017-3746-2
ORIGINAL ARTICLE
Concomitant external pneumatic compression treatment
with consecutive days of high intensity interval training reduces
markers of proteolysis
Cody T. Haun1 · Michael D. Roberts1,2 · Matthew A. Romero1 · Shelby C. Osburn1 · James C. Healy2 ·
Angelique N. Moore2,4 · Christopher B. Mobley1 · Paul A. Roberson1 · Wesley C. Kephart1 · Petey W. Mumford1 ·
Michael D. Goodlett2,3 · David D. Pascoe1 · Jeffrey S. Martin1,2 Received: 1 July 2017 / Accepted: 18 October 2017
© Springer-Verlag GmbH Germany 2017
Abstract Purpose To compare the effects of external pneumatic
compression (EPC) and sham when used concurrently with
high intensity interval training (HIIT) on performancerelated outcomes and recovery-related molecular measures.
Methods Eighteen recreationally endurance-trained male
participants (age: 21.6 ± 2.4 years, BMI: 25.7 ± 0.5 kg/m2,
VO2peak: 51.3 ± 0.9 mL/kg/min) were randomized to balanced sham and EPC treatment groups. Three consecutive
days of HIIT followed by EPC/sham treatment (Days 2–4)
and 3 consecutive days of recovery (Days 5–7) with EPC/
sham only on Days 5–6 were employed. Venipuncture, flexibility and pressure-to-pain threshold (PPT) measurements
were made throughout. Vastus lateralis muscle was biopsied
Communicated by William J. Kraemer.
at PRE (i.e., Day 1), 1-h post-EPC/sham treatment on Day
2 (POST1), and 24-h post-EPC/sham treatment on Day 7
(POST2). 6-km run time trial performance was tested at PRE
and POST2.
Results No group × time interaction was observed for
flexibility, PPT, or serum measures of creatine kinase (CK),
hsCRP, and 8-isoprostane. However, there was a main effect
of time for serum CK (p = 0.005). Change from PRE in 6-km
run times at POST2 were not significantly different between
groups. Significant between-groups differences existed for
change from PRE in atrogin-1 mRNA (p = 0.018) at the
POST1 time point (EPC: − 19.7 ± 8.1%, sham: + 7.7 ± 5.9%)
and atrogin-1 protein concentration (p = 0.013) at the POST2
time point (EPC: − 31.8 ± 7.5%, sham: + 96.0 ± 34.7%).
In addition, change from PRE in poly-Ub proteins was
significantly different between groups at both the POST1
* Jeffrey S. Martin
jmartin@auburn.vcom.edu
Wesley C. Kephart
wck0007@auburn.edu
Cody T. Haun
cth0023@tigermail.auburn.edu
Petey W. Mumford
pwm0009@auburn.edu
Michael D. Roberts
mdr0024@auburn.edu
Michael D. Goodlett
goodlmd@auburn.edu
Matthew A. Romero
mzr0049@auburn.edu
David D. Pascoe
pascodd@auburn.edu
Shelby C. Osburn
sco004@auburn.edu
1
James C. Healy
jch0040@auburn.edu
School of Kinesiology, Auburn University, Auburn, AL,
USA
2
Department of Cell Biology and Physiology, Edward Via
College of Osteopathic Medicine, Auburn Campus, 910
S. Donahue Drive, Auburn, AL 36832, USA
3
Athletics Department, Auburn University, Auburn, AL, USA
4
College of Human Sciences, Auburn University, Auburn,
AL 36849, USA
Angelique N. Moore
anm0013@tigermail.auburn.edu
Christopher B. Mobley
moblecb@auburn.edu
Paul A. Roberson
par0021@tigermail.auburn.edu
13
Vol.:(0123456789)
(EPC: − 26.0 ± 10.3%, sham: + 34.8 ± 28.5%; p = 0.046)
and POST2 (EPC: − 33.7 ± 17.2%, sham: + 21.4 ± 14.9%;
p = 0.037) time points.
Conclusions EPC when used concurrently with HIIT and
in subsequent recovery days reduces skeletal muscle markers of proteolysis.
Keywords Skeletal muscle · Pneumatic compression ·
Recovery · Proteolysis · Oxidative stress · Endurance
exercise · High intensity interval training
Abbreviations
4HNE4-Hydroxynonenal
AcAcetylated
ANOVAAnalysis of variance
APMHRAge predicted max heart rate
BCABicinchonic acid
BSABovine serum albumin
CKCreatine kinase
ESEffect size
FblFibrillarin
GAPDHGlyceraldehyde 3-phosphate
dehydrogenase
hsCRPHigh sensitivity C-reactive protein
EPCExternal pneumatic compression
GXTGraded exercise testing
GPxGlutathione peroxidase
HIITHigh intensity interval training
IgGImmunoglobulin G
IκBαNuclear factor of kappa B (NF-κB) inhibitor alpha
ILInterleukin
HRHeart rate
MCP-1Monocyte chemoattractant protein-1
MuRF-1Muscle RING finger 1
NSAIDsNon-steroidal anti-inflammatories
NF-κBNuclear factor of kappa B
PGC-1αPeroxisome proliferator-activated receptor
coactivator 1-alpha
poly-UbPoly-ubiquitinated
PPARGC1APeroxisome proliferator-activated receptor
gamma coactivator 1-alpha
PPTPressure-to-pain threshold
SOD2Superoxide dismutase 2
TNF-αTumor necrosis factorα
Introduction
External pneumatic compression (EPC) is a treatment
modality used in both clinical and athletic settings. EPC
involves the inflation of cuffs and/or sleeves with air at
selected pressures which generally cover the upper or lower
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Eur J Appl Physiol
extremity limbs. Historically, EPC has been successfully
employed in clinical settings to combat lymphedema by
increasing lymph transport from limbs and decreasing associated pain from swelling (Muluk et al. 2013; Partsch et al.
2008). However, EPC use for recovery purposes in athletic
settings has been increasingly employed and investigated.
While some investigations have indicated little to no positive effect of EPC on performance (Cochrane et al. 2013;
Martin et al. 2015b, c; Overmayer and Driller 2017) and
skeletal muscle glycogen re-synthesis (Keck 2015), several
other investigations have reported positive findings which
may be pertinent to the recovery-adaptation response when
used concurrently with training.
We recently performed an exploratory study examining
the effect of EPC on functional and molecular outcomes
when used concurrently with consecutive bouts of voluminous resistance exercise (Haun et al. 2017). Briefly, that
sham-controlled investigation involved daily EPC use with
heavy back squat exercise occurring for three consecutive
days followed by three additional days of recovery with EPC
treatment only. Similar to the findings of Sands et al. (2014,
2015), we found that EPC treatment attenuated decreases
in the pressure-to-pain threshold (PPT) and improved joint
range of motion (i.e., flexibility) outcomes compared to sham
(Haun et al. 2017). Given that increased muscle soreness is
associated with reduced running economy (Braun and Dutto
2003), EPC-mediated attenuation of muscle soreness when
used concurrently with endurance training may be beneficial
from a metabolic (i.e., reduced anaerobic reliance at relative workloads) and/or risk of injury (Cheung et al. 2003)
standpoint. In addition, in the aforementioned EPC and
resistance training study, we observed significantly lower
levels of surrogates of proteolysis and oxidative stress, total
poly-ubiquitinated (poly-Ub) proteins and 4-hydoxynonenal
(4HNE), in muscle tissue biopsied from the vastus lateralis
with EPC compared to sham (Haun et al. 2017). While the
role of oxidative stress in the recovery-adaptation response
is controversial (Urso and Clarkson 2003), the implications
of a reduction in muscle breakdown are apparent. Finally,
we have also previously shown that acute treatment with
EPC, independent of exercise, (1) upregulates peroxisome
proliferator-activated receptor coactivator 1-alpha (PGC-1α)
mRNA in compressed muscle tissue (Kephart et al. 2015)
and (2) has a large effect on PGC-1α localization in skeletal
muscle cell nuclei (Martin et al. 2016a). PGC-1α is known
to be activated by endurance exercise, and is associated with
mitochondrial biogenesis and the skeletal muscle endurance
phenotype (Baar 2004). Notwithstanding, it remains to be
determined if EPC (1) has similar effects in subjects engaged
in intense endurance training to those observed when used
concurrently with resistance training and (2) has an additive/synergistic effect with known myocellular responses to
endurance exercise (e.g., PGC-1).
Eur J Appl Physiol
As a follow-up to our EPC and resistance training
investigation, the purpose of this study was to explore the
effect(s) of EPC on functional, humoral, and myocellular
outcomes when used concurrently with consecutive bouts
of intense endurance training. To this end, we chose to
investigate the effects of EPC compared to sham when
used during and after three consecutive days of high-intensity interval training (HIIT) in recreationally endurancetrained persons. Specifically, we sought to determine the
(1) change in functional measures of muscle soreness and
flexibility and serum measures of muscle damage, oxidative stress and inflammation across three consecutive
days of HIIT and three additional days of recovery with
EPC compared to sham; (2) acute effects of a single bout
of HIIT followed by EPC on endurance exercise-related
skeletal muscle gene expression and protein concentrations
compared to a single bout of HIIT followed by sham; and
(3) change in 6-km run performance and endurance exercise related skeletal muscle gene expression and protein
concentration after three consecutive days of HIIT and
three additional days of recovery with daily EPC treatment
compared to daily sham treatment.
Methods
Fig. 1 Time and events for the study protocol. Prior to initiation of
study procedures, all participants completed a graded exercise test
(GXT) to characterize heart rate responses and VO2peak. 1-week thereafter participants returned for what is termed as Day 1 (i.e., PRE)
where the following baseline measurements/collections were performed: venipuncture, right knee range of motion in a modified lung
position (i.e., flexibility), pressure-to-pain threshold in the right vastus lateralis (i.e., muscle soreness), biopsy of the left vastus lateralis
and 6-km time-trial performance. Participants then reported to the
laboratory ~ 1-week later (i.e., Day 2; POST1) and performed high
intensity interval training (HIIT) immediately followed by randomization to either an external pneumatic compression (EPC) or sham
treatment group and subsequent 1-h treatment. 1-h following treatment on Day 2, a second biopsy of the left vastus lateralis was performed. On the next two consecutive days (Day 3 and 4) venipuncture
was performed followed by flexibility and muscle soreness measures.
Thereafter, participants completed the same HIIT followed by respective treatment protocol. On the next two consecutive days (Days 5–6)
venipuncture and the muscle soreness and flexibility assessments
were performed and were followed by treatment according to group
assignment (no HIIT was performed). Finally, on Day 7 (i.e., POST2)
participants reported to the lab for venipuncture, muscle soreness and
flexibility assessments, a third biopsy of the left vastus lateralis, and a
6-km run time trial performance
Participant characteristics
Prior to initiating this study, the protocol was reviewed and
approved by the Auburn University Institutional Review
Ethics Committee, and was in compliance with the Helsinki
Declaration. All participants gave their informed consent
prior to their inclusion in the study. Apparently healthy
males (N = 18) volunteered to take part in this investigation and completed the Physical Activity Readiness Questionnaire as well as a health history questionnaire to detect
potential risk factors that might be aggravated by strenuous
physical activity. All participants were considered endurance-trained, participating in ≥ 3 days per week of endurance exercise for at least 3 months.
Experimental protocol
Figure 1 provides an outline of the experimental protocol.
For all visits, participants were asked to report following
a 4-h fast and at the same time of day (± 1-h) to control
for metabolic and diurnal variation influence, respectively,
13
on study outcomes. In addition, participants were asked to
forgo any strenuous activity for at least 48-h prior to graded
exercise testing (GXT) and Day 1 procedures and to refrain
from all strenuous activity (except as indicated) throughout the rest of the protocol. Finally, participants were asked
to maintain their habitual dietary and sleep habits, not use
any “recovery” aids (e.g., ice, topical analgesics, etc.), and
to abstain from taking aspirin or non-steroidal anti-inflammatories (NSAIDs) throughout the study protocol, which is
described in detail below.
Graded exercise testing
All participants were initially assessed for heart rate (HR)
responses and VO2peak during a modified Naughton GXT
protocol. Participants were allowed to warm-up by walking
on the treadmill (Woodway ELG; Woodway USA, Waukesha, WI USA) at 4.8 km/h (3.0 mph) for 5 min prior to GXT.
Immediately after the warm-up was completed, the treadmill
incline was set to 1% and speed was set to the participant’s
self-selected comfortable 10-km run pace. Thereafter, the
treadmill grade increased by 2% every 2 min until the participant indicated volitional fatigue. Expired gases during
GXT were continuously analyzed using a TrueMax 2400
metabolic measurement system (ParvoMedics, Sandy, UT,
USA), averaged in 20-s intervals, and the highest 20-s average for VO2 was denoted as the VO2peak. Following GXT,
participants were dismissed and asked to report back in
~ 1-week.
Day 1 [pre‑testing (PRE)]
For the next visit (Day 1 (PRE)], first, venous blood samples
were collected in a 5 mL serum separator tube and a 3 mL
EDTA tube (BD Vacutainer, Franklin Lakes, NJ, USA) for
subsequent analysis of serum and plasma markers, respectively. Then, a baseline (PRE) percutaneous skeletal muscle
biopsy from the left vastus lateralis was performed at a site
midway between the patella and iliac crest using a 6 mm
gauge Bergstrom needle (Product #72-2300506, Millenium
Surgical Corp., Narberth, PA USA) with suction and sterile laboratory procedures as described previously (Martin
et al. 2016b). Approximately 50 mg of tissue was immediately placed in a 1.7 mL polypropylene tube containing
500 µL of cell lysis buffer (Cell Signaling, Danvers, MA,
USA) with pre-added protease and phosphatase inhibitors
and processed for protein analyses as described below. Additionally, 10–20 mg of muscle was placed in a 1.7 mL polypropylene tube containing 500 µL of Ribozol (Ameresco,
Solon, OH USA) for mRNA analyses as described below,
and the remaining tissue was snap-frozen in liquid nitrogen and subsequently stored at − 80 °C. Following biopsy,
flexibility was assessed by measuring knee range of motion
13
Eur J Appl Physiol
during a modified kneeling lunge similar to the methods
described by MacDonald et al. (2013). Briefly, participants
were positioned in the modified lunge position (upright and
erect torso, left knee in line with left ankle—perpendicular
to floor, right knee in contact with floor behind the torso to
the point of stretch—induced discomfort in the right hip).
The right hip angle was measured with a HiRes™ plastic
360° goniometer (­ Baseline®, 12-1000) and this angle was
used for subsequent (i.e., Days 3–7) measurements of flexibility. After positioning, the participant’s right knee was
passively flexed by an investigator until the participant verbally noted the point of discomfort. The right knee angle, in
degrees, at this point of stretch was recorded with a goniometer using the lateral malleolus and lateral epicondyle along
with the center of the vastus lateralis as landmarks. This
procedure was repeated for duplicate measurements after
1 min of passive rest. The observed coefficient of variation
for duplicate measures of right knee angle across all days
was 3.9%. After flexibility assessment, muscle soreness was
measured by applying focal pressure to proximal, medial,
and distal targeted areas of the right vastus lateralis using
an instrumented algometer (Force Ten FDX, Wagner Instruments, Greenwich, CT, USA). Markings were made for each
site with permanent marker and remained visible throughout the duration of the study for each subject. Pressure was
applied at a rate of approximately 5 N/s at each site until the
subject indicated that the pressure became painful. The point
at which the pressure became painful (audibly indicated by
participants) was termed the PPT and the value in N was
recorded. PPT measurements were made sequentially in
cycles from the proximal to medial to distal site three times
for triplicate measures. The average of the triplicate measures at each site was calculated as the respective site PPT
and the average of all measures was calculated as the 3-site
PPT. The observed coefficient of variation for triplicate PPT
measures across all sites and days was 9.4%. Notably, the
digital display of the algometer indicating force of application was blinded to the participants. Finally, to conclude the
Day 1 (PRE) visit, participants completed a 6-km run time
trial on the treadmill (Woodway ELG) at an incline of 1%.
Participants were instructed to complete the 6-km run in the
shortest possible time and were allowed to control their own
speed during the trial. However, participants and investigators were blinded to all data (e.g., time, speed) except for
distance covered.
Days 2–4 (endurance training and treatment bouts)
Approximately 1 week following Day 1 (PRE), participants
reported at the same time of day for Day 2 (POST1) procedures. Upon arrival to the laboratory, participants were
prepared for and completed HIIT. HIIT was 22.5 min in
duration with 15 rounds of running for 45 s and walking for
Eur J Appl Physiol
45 s (1:1 ratio). Briefly, for each interval participants ran
on the treadmill (Woodway ELG) at an incline of 1% and
a speed resulting in a HR equivalent to 90% of their agepredicted max HR (APMHR; 220-age) for 45 s followed by
45 s of rest (i.e., slow walk on treadmill). Treadmill speed
was adjusted as needed for each interval to maintain a HR
equivalent to 90% of APMHR ± 3%. HR was monitored
continuously using a Polar HR monitor (T31, Polar Electro Inc., Lake Success, NY, USA). Immediately following
completion of the exercise bout, participants were randomly
assigned to either a 1-h treatment with EPC (­ NormaTec™
Pro, Newton, MA, USA) or no compression (sham) whereby
the participants had the leg sleeve on but not inflated for 1 h.
The EPC device, described previously in detail (Haun et al.
2017; Martin et al. 2015c, 2016a), consists of two separate
“leg sleeves” which contain five circumferential inflatable
chambers (arranged linearly along the limb) encompassing the leg from the feet to the hip/groin. The “leg sleeves”
are connected to an automated pneumatic pump at which
target inflation pressures for each zone and the duty cycle
can be controlled. However, the unit is commercially marketed with pre-programmed defaults for the duty cycle and
recommended inflation pressure settings. In this study,
we chose to use an inflation protocol consisting of target
inflation pressures of ~ 70 mmHg for each chamber and a
duty cycle that included 30 s of compression in each zone
followed by a 30-s rest period during which all zones are
deflated. 1 h following the conclusion of EPC or sham treatments, participants donated a second muscle biopsy from
the left vastus lateralis (termed POST1) using techniques
and processing procedures described above. The timing of
the POST1 biopsy (1-h post EPC, 2-h post HIIT) was chosen
(1) in alignment with our previous work on EPC and resistance training (Haun et al. 2017) and (2) based on previous
research which demonstrated significant alteration of biopsy
derived molecular measures of mRNA, including PGC-1a,
following a single EPC treatment alone 1-h post-treatment
(Kephart et al. 2015).
For Days 3–4, 24-h following the previous visit, venipuncture and assessment of flexibility of the right leg and the
PPT in the right vastus lateralis was performed as described
above. Participants then performed the HIIT described during Day 2 and were treated for 1 h with either EPC or sham.
Days 5–6 (treatments only)
For Days 5–6 (which occurred after the three consecutive
HIIT sessions), venous blood sampling and assessment of
flexibility of the right leg and the PPT in the right vastus
lateralis was performed via methods described above. Thereafter, participants were again treated for 1 h with either EPC
or sham, but no HIIT training was performed.
Day 7 (post‑testing)
Participants reported 24 h after Day 6 for post-testing (Day
7). Venipuncture and assessment of flexibility of the right
leg and the PPT in the right vastus lateralis was performed
via methods described above. Next, a final biopsy from the
left vastus lateralis was obtained (POST2), and tissue was
processed for protein and RNA analyses as described above.
The timing of the POST2 biopsy was chosen (1) in alignment with our previous work on EPC and resistance training (Haun et al. 2017) and (2) in an effort to capture the
cumulative effects of the experimental conditions, but not
the acute effect of the last HIIT session or EPC/sham treatment. Notably, each biopsy was obtained slightly proximal
to the previous one as this sampling sequence may prevent
variability due to inflammatory signaling that can occur with
multiple biopsy sampling (Van Thienen et al. 2014). Finally,
a 6-km time-trial run was completed as described above.
Experimental procedures
Serum and plasma analyses
On the days of blood collection (Day1/PRE, Days 3–7),
serum and plasma tubes were centrifuged at 3500×g for
5 min at room temperature. Aliquots were then placed in
1.7 mL microcentrifuge tubes and stored at − 80 °C until
batch-processing. Human enzyme linked immunosorbent
assay kits were used to determine serum concentrations
of 8-isoprostane (Item # 516351; Cayman Chemical, Ann
Arbor, MI, USA) and plasma levels of C-reactive protein
(CRP) (Catalog # 11190; Oxis International Inc., Foster
City, CA, USA). An activity assay was used to determine
serum levels of CK (Catalog # 3460-07; Bioo Scientific,
Austin, TX, USA). All kits were performed according
to manufacturer’s instructions, individual samples were
assayed at least in duplicate, and plates were read using a
96-well spectrophotometer (BioTek, Winooski, VT, USA).
In our hands, the coefficient of variation for duplicate/triplicate samples was 8.9, 5.1, and 6.4% for 8-isoprostane,
CRP, and CK, respectively. Of note, venipuncture was not
performed for serum measures at Day2 (POST1) based on
marginal responses at this time point previously observed in
the laboratory (unpublished data).
RNA expression analyses
Immediately following muscle extraction, samples were
homogenized in 500 µL of Ribozol (Ameresco) and stored
at − 80 °C for batch processing. During batch processing,
total ribonucleic acid (RNA) isolation occurred according
to manufacturer’s instructions. RNA concentrations were
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Eur J Appl Physiol
subsequently assessed using a NanoDrop Lite (Thermo
Scientific, Waltham, MA, USA) prior to cDNA synthesis
for mRNA analyses. cDNA was reverse transcribed from
1000 ng of total RNA for real time PCR analyses using
a commercial cDNA synthesis kit (Quanta Biosciences,
Gaithersburg, MD, USA). Real-time PCR was performed
using SYBR-green-based methods with gene-specific primers designed using an online primer designer tool (Primer3Plus, Cambridge, MA, USA). Fold-change values within
each subject from the PRE biopsy were performed using
the Livak method (Schmittgen and Livak 2008), where
− ΔΔCT = (post-treatment gene of interest − post-treatment
geometric mean of GAPDH and Fbl) − (pre-treatment gene
of interest − pre-treatment geometric mean of GAPDH and
Fbl). Following the PCR reaction for each gene, melt curve
analyses were performed to ensure that one PCR product
was amplified per reaction. A list of targeted markers and
PCR primers is listed in Table 1.
Western blotting analyses
Immediately following muscle extraction, samples were
homogenized using a tight-fitting micropestle in cell lysis
buffer described above, insoluble proteins were removed
with centrifugation at 500×g for 5 min at 4 °C, and supernatants containing muscle tissue homogenate were collected
and stored at − 80 °C. After all participants finished the
study, muscle tissue homogenates were batch-assayed for
total protein content using a bicinchonic acid (BCA) Protein
Assay Kit (Thermo Scientific, Waltham, MA, USA).
Cell lysis homogenates were prepared for Western blotting using 4× Laemmli buffer at 1 µg/µL. Thereafter, 20 µL
of prepped samples were loaded onto 12% SDS–polyacrylamide gels (BioRad, Hercules, CA, USA) and subjected to
electrophoresis (180 V @ 60 min) using 1× SDS–PAGE
running buffer (Ameresco). Proteins were then transferred
to polyvinylidene difluoride membranes (BioRad), Ponceau stained and imaged to ensure equal protein loading
between lanes. Thereafter, membranes were blocked for 1 h
at room temperature with 5% nonfat milk powder. Mouse
anti-pan IkBα (1:1,000; Cell Signaling, Catalog #4814),
rabbit anti-pan p65/NF-κB (1:1000; Cell Signaling; Catalog
#8242), rabbit anti-Fbx32 (atrogin-1; 1:500; Abcam, Cambridge, MA, USA; Catalog #ab74023), rabbit anti-MuRF-1
(1:1000; Abcam; Catalog #ab172479), rabbit anti-Ubiquitin
(poly-Ub, 1:1000, Cell Signaling; Catalog #3933), rabbit
anti-4HNE (1:1000; Abcam; Catalog #ab46545), rabbit
anti-catalase (1:1000; GeneTex, Irvine, CA, USA; Catalog
#GTX110704), rabbit anti-GPx (1:1000, Abcam; Catalog
#GTX116040), and rabbit anti-SOD2 (1:2000, Abcam; Catalog #GTX116093) were incubated with membranes overnight at 4 °C in 5% bovine serum albumin (BSA), and the
following day membranes were incubated with respective
horseradish peroxidase-conjugated anti-rabbit (1:2000, Cell
Signaling; Catalog #7074) or anti-mouse IgG (1:2000, Cell
Signaling, Catalog #7076) or at room temperature for 1 h
prior to membrane development. Membrane development
Table 1 Primer sequences for real time PCR
Gene
Inflammation
IL-6
MCP-1
TNF-α
Metabolism
PPARGC1A
Proteolysis
Atrogin-1
MuRF-1
Redox status
Catalase
GPx
SOD2
Housekeeping genes
Fbl
GAPDH
Forward primer (5′ → 3′)
Reverse primer (5′ → 3′)
AGG​AGA​CTT​GCC​TGG​TGA​AA
TCC​CAA​AGA​AGC​TGT​GAT​CTTCA
TCC​TTC​AGA​CAC​CCT​CAA​CC
CAG​GGG​TGG​TTA​TTG​CAT​CT
CAG​ATT​CTT​GGG​TGG​AGT​GA
AGG​CCC​CAG​TTT​GAA​TTC​TT
CAA​GCC​AAA​CCA​ACA​ACT​TTA​TCT​CT
CAC​ACT​TAA​GGT​GCG​TTC​AAT​AGT​C
ATG​TGC​GTG​TAT​CGG​ATG​G
GCC​TTC​TTC​GCC​TTC​TCC​
AAG​GCA​GGT​CAG​TGA​AGC​
AGC​TCA​TAC​AGA​CTC​AGT​TCC
CTG​ACT​ACG​GGA​GCC​ACA​TC
ACG​AGG​GAG​GAA​CAC​CTG​AT
GTT​GGG​GTT​GGC​TTG​GTT​TC
AGA​TCC​GGA​CTG​CAC​AAA​GG
TCT​GGC​AGA​GAC​TGG​GAT​CA
GCC​TGT​TGT​TCC​TTG​CAG​TG
CCC​ACA​CCT​TCC​TGC​GTA​AT
AAC​CTG​CCA​AAT​ATG​ATG​AC
GCT​GAG​GCT​GTG​GAG​TCA​AT
TCA​TAC​CAG​GAA​ATG​AGC​TT
All primers were designed using PrimerPlus3 (Cambridge, MA, USA) and BLASTed against other potential mRNA targets using the online
NCBI Nucleotide database (Bethesda, MD). IL-6, interleukin-6, MCP-1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor α;
PPARGC1A, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; MuRF-1, muscle RING finger 1; GPx, glutathione peroxidase, SOD2, superoxide dismutase 2; Fbl, fibrillarin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase
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Eur J Appl Physiol
was performed using an enhanced chemiluminescent reagent (Luminata Forte HRP substrate; Millipore, Billerica,
MA, USA), and band densitometry was performed through
the use of a gel documentation system and associated densitometry software (UVP, Upland, CA, USA). Of note, the
densitometry values for all protein targets were normalized
to Ponceau densities.
Immunoprecipitation for determination of acetylated
PGC‑1α
To measure acetylated (Ac) PGC-1α levels, 200 µL of cell
lysate was incubated with rabbit anti-acetylated lysine
antibody (1:1000, Cell Signaling; Catalog #9441) overnight at 4 °C. The following day, protein A agarose beads
(20 μL of 50% bead slurry, Cell Signaling) were added to
antibody–lysate mixtures and rocked 3 h at 4 °C. Samples
were then microcentrifuged for 30 s at 4 °C, supernatants
were discarded, and the pellet was washed with 500 µL of
cell lysis buffer (recipe described above, Cell Signaling).
After two additional repeat washes, samples were microcentrifuged for 30 s at 4 °C, supernatants were discarded,
beads were resuspended with 20 µL of 4× Laemmli buffer,
and samples were heated at 100 °C for 5 min. Thereafter,
samples were loaded onto 12% SDS–polyacrylamide gels
(BioRad, Hercules, CA, USA) and subjected to electrophoresis and transfer as described above. Membranes were
incubated with rabbit anti-PGC-1α (1:1000; Abcam, Catalog #ab54481) overnight, horseradish peroxidase-conjugated
anti-rabbit IgG (1:2000, Cell Signaling, Catalog #7074) at
room temperature for 1 h prior to membrane development,
and developed as described above. The densitometry values for Ac-PGC-1α were normalized to Ponceau densities,
and these values were normalized to PRE values to obtain
fold-change values from 1.00. Ac-PGC-1α raw density values were also normalized to raw PGC-1α values to obtain
Ac-PGC-1α/PGC-1α ratios.
Statistics
For all statistical analyses, an alpha level of p ≤ 0.05 was
required for statistical significance. Independent t tests were
performed for between-groups (EPC vs. sham) comparisons
of age, height, weight, BMI, and VO2peak at study entry.
Respective to the primary aims of the present study, (1) to
evaluate change in muscle soreness, flexibility and serum
measures for all sampled time points with EPC compared to
sham 2 × 6 [group (EPC, sham) × time (PRE and Days3–7)]
repeated measures ANOVAs were performed using absolute
values; (2) to compare the acute effects of a single bout of
HIIT followed by EPC and sham on change in skeletal muscle markers, between groups Welch–Satterthwaite independent t tests on change from PRE at POST1 were performed,
and (3) to compare the change in skeletal muscle markers
and 6-km run performance 24 h after three consecutive days
of HIIT and three additional days of recovery with daily EPC
treatment compared to daily sham treatment, Welch–Satterthwaite independent t tests were performed on change from
PRE at POST2 were performed. For ANOVAs, Mauchly’s
test of sphericity was performed to assure equal variances
and normally distributed data. In the event that sphericity
was not met, Huynh–Feldt correction was applied to hypothesis testing. When a significant main effect of time (withinsubjects factor) was observed, each time point was compared
to PRE using Student’s paired t tests. In these instances,
Bonferroni adjustments were applied (α/5 = 0.01). Grouped
data are presented as mean ± standard error and data respective to effect sizes/post-hoc comparisons are presented as
the mean difference and [95% confidence interval (lower
limit, upper limit)]. All statistics were performed using SPSS
v22.0 (Chicago, IL, USA).
Results
Participant characteristics
Participant characteristics are presented in Table 2. No significant between-group differences were observed for age,
height, body mass, body mass index, or VO2peak (p > 0.05).
Functional outcomes
Data regarding effects of EPC and sham on functional outcomes are presented in Fig. 2. For muscle soreness (i.e.,
PPT) a significant main effect of time, but no main effect of
group or time*group interaction, was observed at all sites as
well as for the 3-site average (Fig. 2a–d). Across both treatment groups, the PPT was significantly reduced relative to
PRE at Day 3 [− 16.5 N (− 8.8, − 24.2); p < 0.001] and Day
5 [− 13.5 N (− 5.4, − 21.6); p = 0.003] for the 3-site average.
No main effects or interaction were observed for changes in
flexibility (i.e., modified kneeling lunge knee angle; Fig. 2e).
Finally, change from PRE at POST2 in 6 km time trial performance was not significantly different between groups
(Fig. 2f).
Blood markers of muscle damage, oxidative stress
and inflammation
Serum CK activity, a marker of muscle damage, demonstrated a main effect of time but no main effect of group
or group × time interaction (Fig. 3a). Across both groups,
serum CK was significantly higher relative to PRE at Day 3
[+ 38.7 U/L (+ 22.5, + 54.9); p < 0.001], Day 4 [+ 57.2 U/L
(+ 16.8, + 97.5); p = 0.009], and Day 5 [+ 32.1 U/L (+ 14.6,
13
Eur J Appl Physiol
Table 2 Participant
characteristics
Age (years)
Height (m)
Body mass (kg)
BMI (kg/m2)
VO2peak (mL/kg/min)
Overall (n = 18)
EPC (n = 9)
Sham (n = 9)
p value
(EPC vs.
sham)
21.1 ± 0.4
1.79 ± 0.02
82.4 ± 1.6
25.7 ± 0.5
51.3 ± 0.9
21.0 ± 0.4
1.79 ± 0.03
80.1 ± 2.2
24.9 ± 0.7
51.5 ± 1.5
21.1 ± 0.6
1.79 ± 0.03
85.0 ± 2.2
26.5 ± 0.5
51.0 ± 0.9
0.872
0.952
0.130
0.093
0.785
Values are mean ± sem. p values from between-groups intendent t tests
BMI body mass index; VO2peak peak 30-s average VO2 observed during graded exercise testing
+ 49.6); p = 0.001]. For 8-isoprostane (Fig. 3b), a marker of
lipid peroxidation, and hsCRP (Fig. 3c), a marker of inflammation, no significant main effects or time × group interactions were observed.
Skeletal muscle gene expression
Targeted gene expression responses to the protocol herein
are presented in Table 3. Change in atrogin-1 gene expression from PRE were significantly different between
groups at the POST1 time point [mean difference: − 0.28
(− 0.19, − 0.37); p = 0.018], but not the POST2 time point
(p = 0.313). Atrogin-1 mRNA levels decreased 19.7 ± 8.1%
from PRE at the POST1 time point in the EPC group and
increased 7.7 ± 5.9% in the sham group. No other genes were
found to be significantly different between groups at either
the POST1 or POST2 time point.
and + 21.4 ± 14.9% at POST1 and POST 2, respectively, in
the sham group (Fig. 4c).
Discussion
We consider the following as primary findings of this investigation: (1) no significant differences existed between
groups in functional variables related to exercise performance (e.g., flexibility, soreness, 6-km run time) or serum
measures of muscle damage, oxidative stress and inflammation, (2) compared to sham, 1-h of EPC treatment following
a single bout of HIIT was associated with lower atrogin-1
gene expression and poly-Ub protein concentrations, and
(3) compared to daily sham treatment, daily EPC treatment
was associated with significantly less atrogin-1 and poly-Ub
protein expression 24 h after three consecutive days of HIIT
and three additional days of recovery.
Skeletal muscle protein expression patterns
Functional measures
Targeted protein expression responses to the protocol herein
are presented in Table 4. No significant between-group differences for change from PRE were observed at the POST1
or POST2 time point for inflammation, metabolism, or
redox status related proteins. Change from PRE in atrogin-1
protein expression was significantly different between
groups at the POST2 time point [mean difference: 128%
(36%, 217%); p = 0.013]. Atrogin-1 decreased 31.8 ± 7.5%
from PRE at the POST2 time point in the EPC group and
increased 96.0 ± 34.7% in the sham group (Fig. 4a). No significant between groups differences for change from PRE
were observed at either the POST1 or POST2 time point for
MuRF-1 protein expression (Fig. 4b). Change from PRE in
poly-Ub proteins was significantly different between groups
at the POST1 [mean difference: 60.7% (1.3%, 120.2%);
p = 0.046] and POST2 time points [mean difference: 55.1%
(4.2%, 106.0%); p = 0.037]. Change from PRE in Poly-Ub
proteins was − 26 ± 10.3 and − 33.7 ± 17.2% at the POST1
and POST2, respectively, in the EPC group and + 34.8 ± 28.5
Flexibility, muscle soreness, and 6-km run time trial performance changes were not significantly different between
groups. The lack of an effect on flexibility and muscle soreness, compared to the differences observed in our resistance
exercise study involving EPC, is likely associated with the
greater eccentric component and tissue-damaging forces
involved in heavy resistance exercise (Haun et al. 2017).
Indeed, Nosaka et al. reported significantly worsened maximal isometric force production and resting joint angles along
with significantly greater muscle soreness and plasma CK
activity when subjects completed 12 maximum eccentric
muscle actions of elbow flexion every 15 s for 3 min compared to a group completing 2 h of consistent elbow flexion
with a load corresponding to ~ 10% of maximal isometric
force (Nosaka et al. 2002). Thus, the nature of the exercise, and resultant magnitude of change from baseline in
flexibility and muscle soreness in this study, likely explains
the lack of differences in these parameters and seems to
have prohibited identification of a treatment effect of EPC.
13
Eur J Appl Physiol
Fig. 2 Functional changes in response to EPC and sham throughout
the protocol utilized herein. Muscle soreness [i.e., pressure-to-pain
threshold (PPT)] along the right vastus lateralis at a proximal, medial,
and distal site is presented in a–c and a composite 3-site average is
presented in d. Flexibility assessed via right knee range of motion in
a modified lunge position is presented in e. 6-km run time trial performance is presented in f. For all panels data are presented as mean
fold-change from PRE ± standard error. Repeated measures ANOVAs
for PPT and flexibility measures and between groups independent t
tests for change from PRE in 6 km run time at the POST2 time point
were performed with an α ≤ 0.05 required for statistical significance.
Main effects of time are presented as bold p values. Post-hoc testing for a main effect of time was performed using Student’s paired t
tests and an α ≤ 0.01 was required for statistical significance. *, time
point(s) (collapsed across groups) significantly different from PRE
In addition, on average, 6-km run times were marginally
reduced at POST2 (− 3.5 ± 1.1%) indicating that the protocol
“missed the mark” in terms of evaluating performance at a
time point when it was substantially depressed post-high
intensity training.
structure at the sarcolemma and Z-disk (Brancaccio et al.
2007), serum hsCRP was used as a surrogate of systemic
inflammation given its acute synthesis in defensive or adaptive responses to inflammatory stimuli (Black et al. 2004),
and 8-isoprostane levels served as a proxy of the magnitude of systemic oxidative stress (Nikolaidis et al. 2011).
A significant main effect of time for serum CK levels was
observed which served as confirmation that the HIIT and
protocol utilized herein elicited significant myocellular damage. However, similar to our findings with resistance training
Serum measures
In this investigation, serum CK approximated the magnitude of muscle damage secondary to disruption of myocyte
13
Eur J Appl Physiol
(Haun et al. 2017), this was surprising. However, in both
the sham and EPC groups there was no increase in local (i.e.,
skeletal muscle) 4HNE expression which is in contrast with
our previous findings with resistance training (Haun et al.
2017). Thus, the relatively limited level of induced myocellular oxidative stress we observed herein likely contributes
to these observed findings.
mRNA and protein expression
Fig. 3 Humoral markers of muscle damage, oxidative stress, and
inflammation in response to EPC and sham throughout the protocol
utilized herein. Creatine kinase (CK; a), 8-isoprostane (b), and high
sensitivity C-reactive protein (hsCRP; c) concentrations in the blood
were measured at baseline (PRE/Day1) and on Days 3–7 of the protocol. Data are presented as mean fold-change from PRE ± standard
error. Repeated measures ANOVAs were performed with an α ≤ 0.05
required for statistical significance. Main effects of time are presented
as bold p values. Post-hoc testing for a main effect of time was performed using Student’s paired t tests and an α ≤ 0.01 was required for
statistical significance. *, time point(s) (collapsed across groups) significantly different from PRE
and EPC, there was no statistically significant effect of EPC
treatment on hsCRP and CK responses associated with the
protocol. In addition, no statistically significant group × time
interaction was observed for serum 8-isoprostane concentrations. Given our previous observations of significant reductions in local skeletal muscle markers of oxidative stress
(i.e., 4HNE) when EPC is used with resistance training
13
Interestingly, atrogin-1 mRNA expression was the only
significantly different mRNA expression pattern observed
between groups with relatively lower levels at the POST1
time point in the EPC group compared to sham (Table 3).
Atrogin-1 is an E3 ligase involved in tagging proteins for
degradation in the 20S proteasome core of the ubiquitin/
proteasome pathway (Louis et al. 2007). Although E2 and
E3 ligase gene expression does not appear to correlate with
their activity (Lecker et al. 1999), in this study, the relatively lower atrogin-1 gene expression with EPC treatment
at the POST1 time point was associated with relatively
lower atrogin-1 protein expression at the POST2 time-point
compared to sham (Fig. 4a). Moreover, change from PRE
in poly-Ub proteins at the POST1 and POST2 time points
was significantly different between-groups with an increase
being observed in the sham group compared to a reduction
in the EPC group suggesting a relatively less proteolyticrelated signaling and events. It is thought that the response
to intense endurance exercise of sufficient duration initially involves a decrease in muscle protein synthesis and
an increase in proteolysis (Rennie and Tipton 2000; Kee
et al. 2002). Although the exact mechanism is unclear in
this investigation, we posit that suppression of proteolytic
markers with EPC compared to sham is due to potentiation
of hormone, nutrient and metabolite delivery and clearance
to/from the skeletal muscle tissue mediated by the repeated
compression relaxation cycles occurring along the lower
limbs during EPC treatment. Indeed, perturbations in lymphatic clearance (Vincent et al. 2005), endothelial surface
area for diffusion via arteriolar vasodilation and/or microcirculatory blood flow (Kolka and Bergman 2012), and
alterations in interstitial pressures (Reed and Rubin 2010)
in response to EPC treatment may augment hormone and
nutrient delivery/uptake in the skeletal muscle that can
down-regulate ubiquitin–proteasome catalyzed skeletal
muscle proteolysis (Biolo et al. 1997, 1999; Chotechuang
et al. 2011; Tesseraud et al. 2007). We have previously
noted that skeletal muscle blood flow (Martin et al. 2016a)
and vascular reactivity (Martin et al. 2015a) are increased
with EPC. Moreover, we have also observed significantly
reduced poly-Ub proteins with EPC compared to sham with
voluminous resistance training (Haun et al. 2017). Granted,
we did not investigate the effects of EPC on muscle protein
Eur J Appl Physiol
Table 3 Effects of EPC
following high intensity interval
training on skeletal muscle gene
expression
Marker (s)
Inflammation
IL-6
MCP-1
TNF-α
Metabolism
PPARGC1A
Proteolysis
Atrogin-1
MuRF-1
Redox status
Catalase
GPx
SOD2
Sham-POST1
Sham-POST2
EPC-POST1
EPC-POST2
p values (EPC vs.
sham)
POST1
POST2
1.38 ± 0.31
1.67 ± 0.36
1.06 ± 0.19
1.59 ± 0.30
1.10 ± 0.21
1.65 ± 0.36
1.56 ± 0.33
1.74 ± 0.77
1.05 ± 0.25
1.13 ± 0.27
1.78 ± 0.45
1.17 ± 0.35
0.698
0.937
0.975
0.270
0.202
0.369
2.20 ± 0.40
1.16 ± 0.09
2.48 ± 0.35
0.94 ± 0.11
0.611
0.151
1.08 ± 0.06
1.21 ± 0.24
1.05 ± 0.10
0.95 ± 0.16
0.80 ± 0.08
1.11 ± 0.20
0.90 ± 0.11
1.05 ± 0.21
0.018
0.770
0.313
0.758
1.19 ± 0.09
1.08 ± 0.10
0.88 ± 0.13
1.08 ± 0.08
1.01 ± 0.10
0.97 ± 0.13
1.09 ± 0.14
1.11 ± 0.03
1.22 ± 0.15
1.00 ± 0.16
1.10 ± 0.09
1.11 ± 0.20
0.579
0.783
0.108
0.653
0.495
0.549
All data are expressed as fold change from 1.00 (mean ± standard error, n = 7–9 subjects per target). Statistical comparisons from between-groups independent t tests (significance indicated in bold). Other notes:
Gene abbreviations are in Table 1 which denotes primer sequences employed herein
Table 4 Effects of EPC
following high intensity interval
training on skeletal muscle
protein expression
Marker (s)
Inflammation
pan-IκBα
pan-NF-κB p65
Metabolism
PGC-1α
Ac-PGC-1α
Ac-PGC-1α/ PGC-1α
Proteolysis
Atrogin-1
MuRF-1
Poly-Ub
Redox status
4HNE
Catalase
GPx
SOD2
Sham-POST1
Sham-POST2
EPC-POST1
EPC-POST2
p values (EPC vs.
sham)
POST1
POST2
0.93 ± 0.11
1.01 ± 0.11
0.83 ± 0.10
0.74 ± 0.10
0.85 ± 0.15
0.85 ± 0.18
1.03 ± 0.10
0.97 ± 0.15
0.702
0.452
0.568
0.233
1.36 ± 0.15
2.02 ± 0.64
1.17 ± 0.47
1.15 ± 0.07
1.41 ± 0.44
1.12 ± 0.41
1.16 ± 0.25
1.75 ± 0.51
0.99 ± 0.32
1.17 ± 0.36
1.24 ± 0.44
1.28 ± 0.41
0.531
0.753
0.760
0.956
0.792
0.784
1.18 ± 0.36
1.35 ± 0.28
1.35 ± 0.29
1.96 ± 0.35
1.60 ± 0.46
1.21 ± 0.15
0.83 ± 0.16
1.29 ± 0.15
0.74 ± 0.10
0.68 ± 0.08
1.16 ± 0.14
0.66 ± 0.17
0.372
0.854
0.046
0.013
0.398
0.037
0.97 ± 0.08
0.84 ± 0.13
1.00 ± 0.29
1.04 ± 0.15
0.85 ± 0.08
1.02 ± 0.10
0.90 ± 0.28
1.07 ± 0.08
1.09 ± 0.08
1.09 ± 0.07
0.92 ± 0.29
1.00 ± 0.10
0.93 ± 0.06
1.16 ± 0.08
0.93 ± 0.40
0.96 ± 0.11
0.287
0.126
0.848
0.853
0.469
0.326
0.942
0.446
All data are expressed as fold change from 1.00 (mean ± standard error, n = 7–9 subjects per target). Statistical comparisons from between-groups independent t tests (significance indicated in bold)
IκBα nuclear factor of kappa B (NF-κB) inhibitor alpha, PGC-1α peroxisome proliferator-activated receptor coactivator 1-alpha, Ac- acetylated, MuRF-1 muscle RING finger-1, poly-Ub poly-ubiquitinated protein,
4HNE 4-hydroxynonenal, GPx glutathione peroxidase, SOD2 superoxide dismutase 2
synthesis rates herein and cannot conclude if the reduction
in markers of proteolysis results in improved skeletal muscle
protein balance. However, the results suggest that EPC could
positively impact recovery-adaptation through reductions in
muscle catabolism in response to training.
Limitations
The present study is not without limitations. First, vastus
lateralis skeletal muscle biopsies were only collected at the
PRE, POST1 and POST2 time points. Thus, the cumulative
13
Fig. 4 Protein expression patterns related to proteolytic signaling. At baseline (PRE), 1 h following high intensity interval training
(HIIT) and treatment with EPC or sham (POST1), and 24 h following three consecutive days of HIIT and treatment with EPC or sham
and two additional, consecutive days of treatment with EPC or sham
(POST2) protein expression patterns related to proteolytic signaling
were probed. Western blot analysis of protein concentrations in vastus
13
Eur J Appl Physiol
lateralis biopsy samples are presented in a atrogin-1, b MuRF-1, and
c poly-ubiqutinated (poly-Ub) proteins. Representative images and
respective Ponceau images for all proteins are presented in d. Values
represent the mean fold change of protein expression normalized to
ponceau from PRE ± standard error. Change from PRE at POST1 and
POST2 were compared using independent t tests. ϕ, significantly different between groups (p < 0.05)
Eur J Appl Physiol
effects of three consecutive days of HIIT training with and
without EPC treatment on skeletal muscle gene expression
and protein concentrations remain unknown. Notably, we
chose to evaluate the acute effects of HIIT and EPC or sham
after the first session given that the physiological response
to repeated exercise on successive days is likely less robust
(McHugh 2003). Second, with respect to the acute effects
of HIIT and EPC or sham, biopsies were only taken at 1-h
post EPC/sham treatment (2-h post HIIT). Thus, in consideration of the timing and transient nature of cellular signaling and gene expression responses, it is likely that we did
not capture all of the signals associated with the observed
physiological response(s). Third, biopsies corresponding to
the PRE time point commenced 1 week prior to the POST1
time point. Therefore, it is possible that some variability in
gene expression and protein concentration existed due to the
time between the PRE and POST1 measures. However, we
elected to use the 1-week of time between PRE and POST1
to (1) allow complete recovery from the initial 6-km run
and (2) limit biochemical artifact secondary to myocellular
disturbances from the biopsy procedure itself (Van Thienen
et al. 2014). Finally, we did not conduct any formal dietary
analysis. Indeed, although participants were instructed to
maintain normal dietary habits throughout the study, without an objective measure of nutritional intake during the
study the potential influence of dietary behaviors cannot be
excluded.
Conclusion(s)
In conclusion, compared to sham, dynamic EPC treatment
of the lower limbs concurrently with HIIT and the protocol
herein reduces markers of proteolysis in skeletal muscle tissue, but not flexibility, soreness, or 6-km time trial performance. More research clarifying the specific mechanisms
whereby EPC reduces markers of oxidative stress and proteolysis is warranted. Additionally, at present, investigations
into the long-term use of EPC in the context of ongoing
training for athletic performance is non-existent. Future
studies can provide insight into the long-term effects of EPC
and allow a better understanding of specific dose–response
relationships of EPC and adaptations influencing exercise
and/or sport performance.
Acknowledgements Partial reagent and participant compensation
costs (50%) were paid through a contract awarded to J.S.M. by Normatec (Newton Centre, MA, USA). The funders did have a role in
study design, but had no role in data collection and analysis, decision
to publish, or preparation of the manuscript. The results of this study
are presented clearly, honestly, and without fabrication, falsification,
or inappropriate data manipulation. The authors wish to thank the participants for their time and compliance with demands associated with
the study protocol.
Compliance with ethical standards Conflict of interest The authors have no conflicts of interest to disclose.
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